Crosslinked human or animal tissue products and their methods of manufacture and use

ABSTRACT

Degradable bioprostheses made of collagen-based material having amine-based and ester-based crosslinks are provided, as are methods for their formation and use. Some embodiments of the present invention are directed towards a method of controlling the ratio of amine-based crosslinks to ester-based crosslinks within a collagen-based material to provide a tailorably crosslinked collagen-based material. Some embodiments provide a method of making a degradable bioprosthesis involving controlling crosslinking to afford a degradable bioprosthesis that is partially crosslinked. By controlling the ratio of amine-based to ester-based crosslinks, by controlling the level of crosslinking, or by controlling both of these features, degradable bioprostheses with tailored degradation rates can be synthesized. Some embodiments of degradable bioprostheses have degradation rates that are tailored to allow their use in particular medical applications. Some embodiments are directed towards methods of use degradable bioprostheses in wound healing, tissue repair, and tissue supplementation.

CROSS-REFERENCE

This application is a continuation of U.S. application Ser. No.14/527,571, filed Oct. 29, 2014, which is a continuation of U.S. patentapplication Ser. No. 13/560,713, filed Jul. 27, 2012, which claims thebenefit of U.S. Provisional Application No. 61/512,801, filed Jul. 28,2011. The contents of all of the above-referenced applications areincorporated by reference herein in their entireties.

BACKGROUND OF THE INVENTION

Field of the Invention

Embodiments of the present invention are directed to controlling acollagen crosslinking process to create crosslinked products withtailorable degradation rates for medical purposes. Further embodimentsare directed to products such as skin substitutes and surgical mesh madefrom such processes, and methods of using such products in medicalprocedures such as treating chronic wounds, supplementing healing,tissue support, and reconstructive surgery.

Description of the Related Art

Many medical products are composed from human or animal tissue-basedmaterials. Examples of these medical products include, for example,heart valves, vascular grafts, urinary bladder prostheses, tendonprostheses, hernia patches, surgical mesh, and skin substitutes. Anillustration of a specific human or animal tissue based product is theheart valve prosthesis. Heart valve prostheses are typically made fromeither porcine aortic valves or bovine pericardium. Such valves aretypically made by pretreating the tissue with glutaraldehyde or othercrosslinking agents and sewing the tissue into a flexible metallic alloyor polymeric stent. These animal tissue starting materials mainlyconsist of collagen, which provides the tissues with their neededmechanical strength and flexibility.

Collagen-based materials, including whole tissue, are finding increaseduse in the manufacture of biomedical devices, such as prostheticimplants. Collagen is a naturally occurring protein featuring goodbiocompatibility. It is the major structural component of vertebrates,forming extracellular fibers or networks in practically every tissue ofthe body, including skin, bone, cartilage, and blood vessels. As anatural component of the extracellular matrix, collagen provides a goodphysiological, isotropic environment that promotes the growth andfunction of different cell types and facilitates rapid overgrowth ofhost tissue in medical devices after implantation.

Basically three types of collagen-based materials can be identified,based on the differences in the purity and integrity of the collagenfiber bundle network initially present in the material. The first typeincludes whole tissue including non-collagenous substances or cells. Asa result of using whole tissue, the naturally occurring composition andthe native strength and structure of the collagen fiber bundle networkare preserved. Whole tissue xenografts have been used in construction ofheart valve prostheses and in many other biomedical prostheses. However,the presence of soluble proteins, glycoproteins, glycosaminoglycans, andcellular components in such whole tissue xenografts may induce animmunological response of the host organism to the implant.

The second type of collagen-based material includes only the collagenmatrix without the non-collagenous substances. The naturally occurringstructure of the collagen fiber bundle network is thus preserved, butthe antigenicity of the material is reduced. The fibrous collagenmaterials obtained by removing the antigenic non-collagenous substanceswill generally have suitable mechanical properties.

The third type of collagen-based material is purified fibrous collagen.Purified collagen is obtained from whole tissue by first dispersing orsolubilizing the whole tissue by either mechanical or enzymatic action.The collagen dispersion or solution is then reconstituted by either airdrying, lyophilizing, or precipitating out the collagen. A variety ofgeometrical shapes like sheets, tubes, sponges or fibers can be obtainedfrom the collagen in this way. The resulting materials, however, do nothave the mechanical strength of the naturally occurring fibrous collagenstructure.

A major problem in the use of collagen-based materials for implantation,and especially whole tissue xenografts in which the donor and recipientare phylogenetically distant, is that these materials are prone to acuterejection. This is a rapid and violent immunological reaction that leadsto the destruction of the xenograft. In order to use collagen-basedmaterials in manufactured medical devices, particularly bioprostheticimplants, their durability and in vivo performance typically need to beprotected from an acute immunological reaction. This can be done bycrosslinking the collagen-based materials to suppress the antigenicityof the material in order to prevent the acute rejection reaction. Inaddition, crosslinking is used to preserve or even improve mechanicalproperties and to enhance resistance to degradation.

Crosslinking can be performed by means of physical methods, including,for example, UV irradiation and dehydrothermal crosslinking. Thesemethods result in a direct, but generally low density crosslinking.Several chemical crosslinking methods for collagen-based materials areknown. These methods involve the reaction of a bifunctional reagent withthe amine groups of lysine or hydroxylysine residues on differentpolypeptide chains or the activation of carboxyl groups of glutamic andaspartic acid residues followed by the reaction with an amine group ofanother polypeptide chain to give an amide bond.

Compared with other known methods, glutaraldehyde (GA) crosslinking ofcollagen provides materials with the highest degree of crosslinking. Itis currently the most frequently used chemical crosslinking reagent forcollagen-based materials. Glutaraldehyde is a dialdehyde. The aldehydeis able to chemically interact with amino groups on collagen to formchemical bonds. This crosslinking agent is readily available,inexpensive, and forms aqueous solutions that can effectively crosslinktissue in a relatively short period. Using GA crosslinking, increasedresistance to biodegradation, reduced antigenicity, and improvedmechanical properties of collagen-based materials can be achieved.Despite improved host acceptance, crosslinking of collagen-basedmaterials using GA has shown to have cytotoxic characteristics, both invitro and in vivo. Also, crosslinking of collagen-based materials usingGA tends to result in stiffening of the material and calcification.

Crosslinking can also be accomplished with diisocyanates by bridging ofamine groups on two adjacent polypeptide chains. In the first step,reaction of the isocyanate group with a (hydroxy)lysine amine groupoccurs, resulting in the formation of a urea bond. Thereafter acrosslink is formed by reaction of the second isocyanate group withanother amine group. Diisocyanates do not show condensation reactions asobserved in GA crosslinking. Also, no residual reagents are left in thematerial. A disadvantage, however, is the toxicity of diisocyanates andlimited water solubility of most diisocyanates.

Another method of crosslinking involves the formation of an acyl azide.The acyl azide method involves the activation of carboxyl groups in thepolypeptide chain. The activated groups form crosslinks by reaction withcollagen amine groups of another chain. First, the carboxyl groups areesterified by reaction with an alcohol. This ester is then converted toa hydrazide by reaction with hydrazine (H₂N—NH₂). Acyl azide groups areformed by reaction with an acidic solution of sodium nitrite. At lowtemperatures and basic pH values, the acyl azide group reacts with aprimary amine group to give amide bonds. This multi-step reactionresults in good material properties; however, long reaction times (e.g.,7 days) are necessary. Alternatively, a method has recently beendeveloped that does not need an esterification step or the use ofhydrazine. In this method, a carboxyl group is converted to an acylazide group in one single step by reaction with diphenylphosphorylazide(DPPA). This increases the reaction rate significantly; however, thereaction is carried out in an organic solvent (e.g., DMF), which isundesirable.

Also, water-soluble carbodiimides can be used to activate the freecarboxyl groups of glutamic and aspartic acid moieties in collagen.Activation of the carboxyl groups with carbodiimides, such as1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide.HCl (EDC), givesO-acylisourea groups. A condensation reaction by nucleophilic attack ofa free amine group of a (hydroxy)lysine residue with urea as a leavinggroup results in formation of an amide crosslink. The O-acylisourea canalso be hydrolyzed or rearranged to an N-acylurea, which is much morestable and will not react to form a crosslink. Addition ofN-hydroxysuccinimide (NHS) prevents this rearrangement, however. In thepresence of NHS, the O-acylisourea can be converted to an NHS activatedcarboxyl group, which also can react with a free amine group to form acrosslink. Addition of NHS increases the reaction rate. Also,crosslinking with EDC and NHS provides collagen material with a highdegree of crosslinking; however, it also results in a material with alow tensile strength.

Yet another crosslinking method uses epoxy compounds to crosslinkcollagen. See, for example, U.S. Pat. No. 4,806,595 (Noishiki et al.),U.S. Pat. No. 5,080,670 (Imamura et al.), U.S. Pat. No. 5,880,242 (Hu,et al.), U.S. Pat. No. 6,117,979 (Hendriks et al.), and U.S. Pat. No.7,918,899 (Girardot et al.). Epoxy compounds (i.e., epoxides) canundergo both acid-catalyzed and base-catalyzed reactions with a numberof functional groups, including amine groups and carboxylic acid groups,under the appropriate conditions. Typically, the crosslinking ofcollagen with epoxides is carried out at basic pH (e.g., pH 8-10) withthe result that crosslinking occurs through the free amine groups of thecollagen.

Common to all of these crosslinking methods is the objective to “fullycrosslink” the collagen (generally regarded as achieving crosslinksamong at least 80% of the collagen molecules) in order to createproducts with low immunogenicity and a high resistance to enzymaticattack by the host body (and therefore very long term durability).However, there remains a need to create products, specificallycollagen-based materials, that have great durability (defined asretaining high strength following implant) but which are intended todegrade during healing such that they are essentially fully dissolvedwhen the healing process is complete.

Despite the wide variety of crosslinking agents available, thedegradation profiles of prosthetic collagen materials currently in usefor general surgical reconstruction fall into only two categories: 1)those prosthetic collagen materials that quickly bioresorb when in use,or 2) those prosthetic collagen materials that last longer than one yearin use and, for all intents and purposes, are non-bioresorbable. Withinthe category of quickly bioresorbed materials, two classes ofbioprosthetic collagen materials dominate. The first of such materialsare collagen-based materials that have not been cross-linked (e.g.native collagen). The second is collagen that is fully crosslinked butwith crosslinks that are hydrolyzable, such as ester-based crosslinks.Generally, quickly bioresorbable collagen materials have a functionalduration of 6 to 8 weeks in normal in vivo conditions (such as in awound or surgical site). The time taken to bioresorb may be even less inmore proteolytic environments such as in chronic wounds such as diabeticfoot ulcers. During biodegradation, these quickly bioresorbablecollagenous materials often prematurely lose strength and otherimportant functional characteristics before the wound is completelyhealed, thereby compromising the long term success of the medicalprocedure.

Non-bioresorbable extracellular collagen matrices are historically fullycross-linked materials with non-hydrolyzable crosslinks. Typicalexamples include collagen that has amine-based crosslinks. Theamine-based crosslinking provides a material that is non-bioresorbablein the in vivo biologic environment. Fully crosslinked extracellularcollagen materials with non-hydrolyzable crosslinks currently tend tolast many years, if not a lifetime, when used for surgical repair orreconstruction. While such materials retain strength during the healingprocess, their long presence can be problematic.

SUMMARY OF THE INVENTION

Given the limitations of current collagen-based biomaterials, certainembodiments of the present application provide methods synthesizingdegradable bioprostheses (or singularly a degradable bioprosthesis),compositions of degradable bioprostheses, products made therefrom, andmethods of using said products and compositions. Some embodimentsprovide a method of making a degradable bioprosthesis. In oneembodiment, a method comprises providing a collagen-based material,exposing the collagen-based material to a first buffered solution with apH between 8.0 to 10.5 (or between about 8.0 to about 10.5) for a firstperiod of time to provide a treated collagen-based material, wherein thefirst buffered solution comprises a concentration of a firstcrosslinking agent, exposing the treated collagen-based material to asecond buffered solution with a pH between 3.0 to 5.5 (or between about3.0 to about 5.5) for a second period of time to provide a tailorablycrosslinked collagen-based material, wherein the second bufferedsolution comprises a concentration of a second crosslinking agent, andisolating the tailorably crosslinked collagen-based material to providea degradable bioprosthesis. In some embodiments, a bioprosthesis made bythe method above is provided.

Another embodiment of a method of making a degradable bioprosthesiscomprises providing a collagen-based material and controllablycrosslinking the collagen so that only a portion of the collagen iscrosslinked. In some embodiments a bioprosthesis made by the methodabove is provided. Other methods of making a biodegradable bioprosthesisare described below.

In some embodiments, a crosslinked collagen-based material is provided.In one embodiment, a crosslinked collagen-based material comprisingCrosslink A and Crosslink B represented by

wherein

indicates collagen strands, R¹ is

R² is

and R³ and R⁴ are independently selected from the group consisting of—(CH₂)_(n)— and —(O(CH₂)_(n))_(m)—, where n and m are independently aninteger from 1-6, and the amount of free amines (—NH₂) on the collagenstrands is between 50% and 85% (or between about 50% and about 85%) isprovided. Other methods of making a biodegradable bioprosthesis aredescribed below.

Some embodiments provide a degradable bioprosthesis comprising acrosslinked collagen-based material comprising Crosslink A and CrosslinkB:

wherein

indicates collagen strands, R¹ is

R² is

and R³ and R⁴ are independently selected from the group consisting of—(CH₂)_(n)— and —(O(CH₂)_(n))_(m)—, where n and m are independently aninteger from 1-6, and the amount of free amines (—NH₂) on the collagenstrands is between 50% and 85% (or between about 50% and about 85%) isprovided.

Some embodiments provide a degradable bioprosthesis comprising atailorably crosslinked collagen-based material. In one embodiment, acollagen-based material comprises collagen strands that further compriseamine-based crosslinks and ester-based crosslinks, and the tailorablycrosslinked collagen-based material has a degradation rate between 0.2%to 1.0% (or between about 0.2% to about 1.0%) per hour when subjected toa pronase digestion assay. Other embodiments of a degradablebioprosthesis are described below.

In another embodiment, a degradable bioprosthesis comprising a biologicskin substitute comprising collagen that is between 20% to 80% (orbetween about 20% to about 80%) crosslinked is provided.

Some embodiments provide a method of treating a tissue defect. In oneembodiment, the method comprises positioning a degradable bioprosthesissuch as described above or further herein at, over, or into the tissuedefect, wherein the degradable bioprosthesis comprises a crosslinkedcollagen-based material having a degradation rate between about 0.2% toabout 1.0% per hour when subjected to a pronase digestion assay.

Some embodiments provide a method of treating a wound. In oneembodiment, the method comprises identifying a patient in need of adegradable bioprosthesis to aid in the healing of a wound, determiningan approximate rate of healing of the wound, selecting a degradablebioprosthesis comprising a tailorably crosslinked collagen-basedmaterial having a degradation rate similar to the rate of healing of thewound, and implanting the degradable bioprosthesis over or into thewound.

In another embodiment, a method of treating a wound comprises providinga biologic skin substitute comprising partially crosslinked collagen andplacing the skin substitute over a wound, wherein the degradation of theskin substitute progresses at or about the same rate as the wound heals.Other embodiments of methods of treatment are described below. Suchmethods may utilize any of the compositions, materials, bioprostheses orother structures described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart depicting an embodiment of the method ofsynthesizing a tailorably crosslinked collagen-based bioprosthesis.

FIG. 2 depicts one embodiment of crosslinked collagen-based materialmade using the process described herein.

FIG. 3 is a chart showing the temperature of shrinkage for Examples 1-7and native collagen.

FIG. 4 is a chart showing the resistance of protease digestion ofExamples 1-7 and native collagen.

FIG. 5 is a chart showing the degradation rates (in % mass loss perhour) of Examples 1-7 and native collagen.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

This disclosure is related to compositions and methods of synthesizingand using tailored crosslinked collagen-based animal or human tissue toafford bioprosthetic devices with tailored degradation rates.

As used herein, the term “collagen-based material” refers to materialsthat have been excised from animal or human tissue, which may or may notbe crosslinked. Depending on the level of processing of natural tissue,collagen-based materials may include collagen, tropocollagen, collagenfibrils, or collagen fibers. Collagen exists as a triple helix of aminoacid chains. These triple helical chains, called tropocollagen, furtherassemble to form collagen fibrils. These collagen fibrils assemble toform collagen fibers.

As used herein, the term “collagen strand” refers to tropocollagen,collagen fibrils and/or collagen fibers. Collagen strands have pendantamine (—NH₂) and carboxylic acid (—COOH) groups which are reactive.These amines and carboxylic acid groups are readily crosslinked betweencollagen strands with various crosslinking agents to form structureswith improved medial properties. Crosslinking can be performed by takingadvantage of pendant reactive groups on the collagen strand.

As used herein, the term “degradation time” refers to the amount of timeit takes for a collagen-based material to completely degrade or todegrade to such an extent that it no longer serves the purpose for whichit was medically intended.

As used herein, the term “diepoxide” refers to a compound that has tworeactive epoxide functionalities. Epoxides have long been used ascrosslinking agents for collagen because, when fully reacted, epoxidecrosslinking reduces the immunogenicity of collagen while improving thephysical properties of the material. Useful diepoxides may include, butare not limited to, glycol diglycidyl ether, glycerol diglycidyl ether,butanediol diglycidyl ether, resorcinol diglycidyl ether, 1,6-hexanedioldiglycidyl ether, ethylene glycol diglycidyl ether, diethylene glycoldiglycidyl ether, triethylene glycol diglycidyl ether, polyethyleneglycol diglycidyl ether, propylene glycol diglycidyl ether, dipropyleneglycol diglycidyl ether, and polybutadiene, diglycidyl ether. An exampleof the diepoxide that can be used to crosslink collagen strands is 1,4butanediol digylcidyl ether (BDDGE).

As used herein, the term “crosslinking agent” may refer to diepoxides orcompounds with three or more pendant epoxide functional groups. Usefulcrosslinking agents may include, but are not limited to, the abovementioned diepoxides, glycerol triglycidyl ether, sorbitol polyglycidylether, polyglycerol polyglycidyl ether, pentaerythritol polyglycidylether, diglycerol polyglycidyl ether, glycerol polyglycidyl ether, andtrimethylolpropane polyglycidyl ether.

An unmet need in the area of wound healing, general surgery, andorthopedic surgery is for a collagen-based material that can be tailoredto degrade at intervals, for example, greater the 8 weeks but of lessthan 1 year. This tailored degradation rate can be made to comport withthe healing cycle of each specific condition. Examples of theseconditions include procedures such as hernia repair, diabetic foot ulcerhealing and orthopedic tendon repairs to name only a few. Embodiments ofthe current invention are targeted towards compositions that havetailorable degradation times.

Some embodiments of the present invention are directed towardscontrolling the ratio of amine-based crosslinks to ester-basedcrosslinks within a collagen-based material to provide a tailorablycrosslinked collagen-based material. The inventors of the presentinvention have surprisingly found that by controlling the pH andreaction time during the crosslinking of collagen-based materials usingepoxides, a tailorably crosslinked collagen-based material can beobtained. While not bound by any particular theory, the inventors havefound that by controlling the pH of the reaction, the ratio ofamine-based crosslinking (Crosslink A) to ester-based crosslinking(Crosslink B) in a collagen-based material can be controlled to afford abioprosthesis with a controllably tailored degradation rate. Asmentioned previously, the amine-based crosslinks form stable bonds.Thus, the higher the ratio of amine-based crosslinks, the lower thedegradation rate of the collagen-based materials. Alternatively, theester-based crosslinks hydrolyze quickly in vivo. Thus, the higher theratio of ester-based crosslinks, the higher the degradation rate of thecollagen-based materials. By manipulating the ratio of amine andester-based crosslinks, the degradation rate can be tailorably adjustedto clinically relevant timeframes.

In some embodiments, the diepoxide BDDGE has been shown to fullycrosslink collagen at a concentration of 4% weight to volume, a pH ofbelow 6, and a reaction time of 160 hours. As used herein, the term “%weight to volume” or “% w/v” refers to the weight of solute (g)/volumeof solution (mL)×100. It is believed that at low pH (e.g., 3.0-5.5 pH),crosslinking primarily occurs through a reaction of carboxylates on thecollagen strand with epoxides on the BDDGE. At low pH, it is believedthat carboxylic acids of the collagen strand are more nucleophilic thanamine groups of the collagen strand. At low pH, some amount ofcarboxylic acid group exists as a carboxylate (—RCOO^(⊖)) which is tosome degree nucleophilic, while the amine group is primarily protonatedto form a primary ammonium (—R′NH₃ ^(⊕)), which is not nucleophilic. Thecarboxylate, therefore, preferentially acts as a nucleophile in areaction with an epoxide crosslinking agent. The resulting functionalgroup is an ester, thus forming an ester-based crosslink (as shownbelow).

Ester-based crosslinks, are readily hydrolyzable and are quicklybioresorbable. Thus, the degradation time of collagen that is fullycrosslinked with ester-based crosslinks is near that of native collagenat around 8 weeks or less. Thus, despite improved immunogenicity overnative collagen, crosslinking through these ester groups does little toslow the degradation rate of native collagen. The degradation rate ofcollagen is measured by a % loss in mass over unit of time measured inhours. One of skill in the art will also recognize that whendiepoxide-based crosslinking is carried out at low pH, some amount ofamine-based crosslinks will likely form, as well as crosslinks that arebased on the reaction of both a carboxylate and an amine with adiepoxide (a hetero-crosslink).

It is also known that crosslinking collagen at 9.2 pH with BDDGE for 160hours will fully crosslink the collagen. While not bound by anyparticular mechanism, it is believed that this crosslinking occursprimarily through the amine groups on the collagen strands, which athigher pH exist to some degree as highly nucleophilic free amines(—R′NH₂). The resulting functional group from the reaction of an aminewith an epoxide is an amine, thus this is an amine-based crosslink.

These amine-based crosslinking lead to a non-bioresorbable material witha degradation rate that exceeds a year in vivo. Because the amine-basedcrosslink is so strong, it does not degrade easily under typical in vivoconditions. One of skill in the art will also recognize that whendiepoxide-based crosslinking is carried out at a high pH, some amount ofester-based crosslinks will likely form, as well as crosslinks that arebased on the reaction of both a carboxylate and an amine with adiepoxide (a hetero-crosslink).

In some embodiments, the reaction time is long enough to ensuresubstantially complete crosslinking of the collagen strands. Thesubstantially complete crosslinking of these collagen strands may beimportant for 1) instilling the crosslinked collagen-based material withgood mechanical properties and 2) reducing the number of pendantunreacted epoxide functional groups bound to the crosslinkedcollagen-based material and therefore lowering cytotoxicity. In someembodiments, reaction times are sufficient to decrease the number ofpendant epoxides to such a degree that the resulting materials arebiocompatible. Thus, embodiments of the current strategy results in acrosslinked material which is highly tailorable and biocompatible.

In some embodiments, the ratio of Crosslink A to Crosslink B can beadjusted by controlling the amount of time a collagen-based material isexposed to a buffered solution of a diepoxide at a first pH, then bycontrolling the amount of time the treated material is exposed to asecond buffered solution of a diepoxide at a second pH.

Embodiments of the present invention provide methods of making adegradable bioprosthesis (for example, an implant or implantable device,or a topically applied wound dressing) comprising collagen-basedmaterial that has been controllably crosslinked and is intentionallydesigned to degrade over a proscribed period of time in the body, withsuch time generally less than one year. In some embodiments, thesemethods also involve partially crosslinking the collagen-based material.Advantageously, these methods yield a material with a predictably, andrepeatedly moderate degree of crosslinking, variable crosslinkstabilities, and a moderate/tailored resistance towards enzymaticdigestion following implantation.

Method of Making Bioprosthesis

Some embodiments provide a method of making a degradable bioprosthesisas shown in FIG. 1. First step 101 involves providing a collagen-basedmaterial. In some embodiments, animal or human tissue is dissected andundergoes a decellularization process to result in the collagen-basedmaterial. In some embodiments, depending on the level of processing ofnatural tissue, collagen-based materials may be collagen, tropocollagen,collagen fibrils, or collagen fibers. In some embodiments thecollagen-based material is excised from the pericardium of an animal ora human.

Step 102 involves exposing the collagen-based material to a firstbuffered solution comprising a first crosslinking agent at a first pHfor a first period of time to provide a treated collagen-based material.In some embodiments, the first pH is high enough to result in crosslinksthat are primarily amine-based. In some embodiments, the first bufferedsolution has a pH between 8.0 to 10.5 (or about 8.0 to about 10.5). Insome embodiments, the pH of the first buffered solution may be from 8.9to 9.5 (or about 8.9 to about 9.5), from 9.0 to 9.4 (or about 9.0 toabout 9.4), or from 9.1 to 9.3 (or about 9.1 to about 9.3). In someembodiments, the pH of the first buffered solution may be 9.2 (or about9.2). The concentration of first crosslinking agent in the firstsolution may be from 1% to 10% (or from about 1% to about 10%) (w/v),from 2% to 8% (or from about 2% to about 8%) (w/v), from 3% to 7% (orabout 3% to about 7%) (w/v), or from 4% to 6% (or about 4% to about 6%)(w/v). In some embodiments, the first crosslinking agent concentrationin the first solution is 4% (or about 4%) (w/v). The first period oftime for the crosslinking reaction depends on the desired level ofcrosslinking, and may be from 0.5 hours to 64 hours (or about 0.5 hoursto about 64 hours). In some embodiments, the first period of time may befrom 1 hour to 60 hours (or about 1 hour to about 60 hours), from 10hours to 50 hours (or about 10 hours to about 50 hours), or from 20hours to 40 hours (or about 20 hours to about 40 hours). In someembodiments the first period of time may be from 0.5 hours to 10 hours(or about 0.5 hours to about 10 hours), from 10 hours to 20 hours (orabout 10 hours to about 20 hours), from 20 hours to 30 hours (or about20 hours to about 30 hours), from 30 hours to 40 hours (or about 30hours to about 40 hours), from 40 hours to 50 hours (or about 40 hoursto about 50 hours), from 50 hours to 60 hours (or about 50 hours toabout 60 hours), or from 60 hours to 64 hours (or about 60 hours toabout 64 hours). In some embodiments, the treated collagen-basedmaterial comprises partially crosslinked collagen strands, and thecrosslinks are primarily amine-based crosslinks.

Step 103 involves exposing the treated collagen-based material to asecond buffered solution comprising a second crosslinking agent at a lowpH for a second period of time to provide a tailorably crosslinkedcollagen-based material. The pH of the second buffered solution is lowenough to result in crosslinks that are primarily ester-based. In someembodiments, the pH of the second buffered solution may be from 3.0 to5.5 (or about 3.0 to about 5.5). In some embodiments, the pH of thesecond buffered solution may be from 4.2 to 4.8 (or about 4.2 to about4.8), from 4.3 to 4.7 (or about 4.3 to about 4.7), or from 4.4 to 4.6(or about 4.4 to about 4.6). In some embodiments, the pH of the secondbuffered solution may be 4.5 (or about 4.5). The concentration of secondcrosslinking agent in the second solution may be from 1% to 10% (or fromabout 1% to about 10%) (w/v), from 2% to 8% (or from about 2% to about8%) (w/v), from 3% to 7% (or about 3% to about 7%) (w/v), or from 4% to6% (or about 4% to about 6%) (w/v). In some embodiments, the firstcrosslinking agent concentration in the first solution is 4% (or about4%) (w/v). The second period of time for the crosslinking may be from100 hours to 160 hours (or about 100 hours to about 160 hours). In someembodiments, the second period of time for the crosslinking may be from100 hours to 170 hours (or about 100 hours to about 170 hours), from 110hours to 160 hours (or about 110 hours to about 160 hours), from 120hours to 150 hours (or about 120 hours to about 150 hours), or from 130hours to 140 hours (or about 130 hours to about 140 hours). In someembodiments, the second period of time for the crosslinking may be from100 hours to 110 hours (or about 100 hours to about 110 hours), from 110hours to 120 hours (or about 110 hours to about 120 hours), from 120hours to 130 hours (or about 120 hours to about 130 hours), from 130hours to 140 hours (or about 130 hours to about 140 hours), from 140hours to 150 hours (or about 140 hours to about 150 hours), from 150hours to 160 hours (or about 150 hours to about 160 hours), or from 160to 170 hours (or about 160 hours to about 170 hours). In someembodiments, the second period of time for the crosslinking may beperformed for a period that exceeds 170 hours.

In some embodiments, the second period of time for the crosslinking isenough to reduce the amount of pendant epoxides to the point that thematerial is no longer cytotoxic.

In some embodiments, the total exposure time to the first and secondbuffered solutions (the total of the first period of time and the secondperiod of time) is such that the resulting tailorably crosslinkedcollagen-based material is substantially fully crosslinked. In someembodiments, the total exposure time will be sufficient to afford amaterial that contains a small enough amount of pendant free epoxidessuch that the material is biocompatible. In some embodiments, the sum ofthe first period of time and the second period of time is from 100.5hours to 110 hours (or about 100.5 hours to about 110 hours), from 110hours to 120 hours (or about 110 hours to about 120 hours), from 120hours to 130 hours (or about 120 hours to about 130 hours), from 130hours to 140 hours (or about 130 hours to about 140 hours), from 140hours to 150 hours (or about 140 hours to about 150 hours), and/or from150 hours to 160 hours (or about 150 hours to about 160 hours). In someembodiments, the sum of the first period of time and the second periodof time is 160 hours (or about 160 hours). In some embodiments, the sumof the first and second periods of time is longer than 160 hours.

Alternatively, the pH of the buffered solutions and reaction times insteps 102 and 103 may be reversed in some embodiments. In someembodiments, step 102 is performed before step 103. In otherembodiments, the step 102 may follow step 103. The collagen-basematerial may be exposed to a crosslinking agent solution with a low pHfirst, and then a second crosslinking agent solution with a high pHsecond. In this case, the first buffered solution has a low pH, whilethe second buffered solution has a high pH.

The crosslinking agents in steps 102 and 103 (the first crosslinkingagent and the second crosslinking agent) may be an epoxide. In someembodiments, the first crosslinking agent and the second crosslinkingagent may be diepoxide. In some embodiments, the first and secondcrosslinking agents are independently selected from the group consistingof glycol diglycidyl ether, glycerol diglycidyl ether, glyceroltriglycidyl ether, and butanediol diglycidyl ether. In some embodiments,the first and the second crosslinking agents are the same. In someembodiments, the first and second crosslinking agents are BDDGE. In someembodiments, the first and second crosslinking agents used in thepresent invention are water soluble, non-polymeric epoxies such aspolyol polyglycidylethers.

Step 104 involves isolating the tailorably crosslinked collagen-basedmaterial to provide a degradable bioprosthesis. The tailorablycrosslinked collagen-based material comprises Crosslink A and CrosslinkB. In some embodiments, the ratio of Crosslink A to Crosslink B is from90:10 to 10:90 (or about 90:10 to about 10:90), from 80:20 to 20:80 (orabout 80:20 to about 20:80), from 70:30 to 30:70 (or from about 70:30 toabout 30:70), from 60:40 to 40:60 (or about 60:40 to about 40:60), or1:1 (or about 1:1). In some embodiments, the concentration of the firstcrosslinking agent in the first solution may be from may be from 1% to10% (or from about 1% to about 10%) (w/v), from 2% to 8% (or from about2% to about 8%) (w/v), from 3% to 7% (or about 3% to about 7%) (w/v), orfrom 4% to 6% (or about 4% to about 6%) (w/v), or 4% (or about 4%). Insome embodiments, the concentration of the second crosslinking agent inthe second solution may be from 1% to 10% (or from about 1% to about10%) (w/v), from 2% to 8% (or from about 2% to about 8%) (w/v), from 3%to 7% (or about 3% to about 7%) (w/v), or from 4% to 6% (or about 4% toabout 6%) (w/v), or 4% (or about 4%).

In some embodiments, the amount of free amines on the crosslinkedcollagen-based material ranges from 50% to 85% (or about 50% to about85%), 60% to 75% (or about 60% to about 75%), from 65% to 70% (or about65% to about 70%), or from 65% to 70% (or about 65% to about 70%),relative to the number of free amines on the collagen-based materialbefore exposing to any crosslinking agent.

Some embodiments provide a method of making a degradable bioprosthesisinvolving controlling the crosslinking to afford a degradablebioprosthesis that is partially crosslinked. In some embodiments, tocreate a partially crosslinked degradable bioprosthesis, thecrosslinking reaction can be slowed down. In some embodiments, forexample, the crosslinking reaction can be slowed by decreasing theconcentration of the crosslinking agent, such as from 10% down to 1% insolution (or about 10% to about 1%). In some embodiments, theconcentration of crosslinking agent can be controlled within the rangefrom 1% to 2% (or about 1% to about 2%), from 2% to 3% (or about 2% toabout 3%), from 3% to 4% (or about 3% to about 4%), from 4% to 5% (orabout 4% to about 5%), from 5% to 6% (or about 5% to about 6%), from 6%to 7% (or about 6% to about 7%), from 7% to 8% (or about 7% to about8%), from 8% to 9% (or about 8% to about 9%), or from 9% to 10% (orabout 9% to about 10%).

In some embodiments, the method comprises combining a crosslinking agentwith the collagen-based material in an aqueous medium at a basic or anacidic pH to react with a predetermined portion of the collagen amine orcarboxyl groups to form a degradable bioprosthesis.

In some embodiments, the crosslinking reaction can be slowed down byreducing the acidity of a crosslinking reaction agent intended to reactwith the collagen carboxyl groups, or reducing the alkalinity of acrosslinking reaction agent intended to react with the collagen aminegroups. In some embodiments, the crosslinking reaction can be sloweddown, for example, by reducing the temperature at which the crosslinkingreaction takes place. In some embodiments, the crosslinking reaction canbe slowed down, for example, by reducing the pressure at which thecrosslinking reaction takes place.

In some embodiments, to create a partially crosslinked degradablebioprosthesis, the length of time the collagen-based material is exposedto the crosslinking reaction agent is reduced. In some embodiments, tocreate a partially crosslinked degradable bioprosthesis, thecollagen-based material is masked by chemical or physical means,allowing exposure of only part of the collagen-based material to thecrosslinking reaction agent. In some embodiments, another way to createa partially crosslinked degradable bioprosthesis is to expose only aportion, or less than all surfaces, of the collagen-based material tothe crosslinking reaction agent.

All of these aforementioned methods to slow the crosslinking reaction orto shorten the reaction time, or to slow, inhibit, limit or preventexposure of the collagen-based material to the crosslinking reactionagent can be used individually, or in any combination to achieve theoptimum material properties.

For example, in some embodiments, reducing the concentration of thecrosslinking agent from 5% to 1% in combination with reducing the pH ofthe crosslinking agent from 10 to 8.5, will substantially slow thecrosslinking reaction. In some embodiments, exposing only one surface orside of the collagen-based material to the crosslinking reaction agentcan slow or inhibit the crosslinking reaction and thereby limit theamount of collagen that is crosslinked.

Further details regarding cross-linking processes and materials relevantto this disclosure are described in U.S. Pat. No. 5,880,242, theentirety of which is hereby incorporated by reference.

Embodiments of the present invention achieve a predetermined degree ofcrosslinking by precise control of the concentration of the crosslinkingagent, the pH of the crosslinking agent, the length of time thecollagen-based material is exposed to the crosslinking agent, and thetemperature at which the collagen-based material is exposed to thecrosslinking agent. One preferred degree of crosslinking would becrosslinking only about 50% of the free amine or carboxyl groups whichwould enable the degradable bioprosthesis to retain sufficientresistance to premature enzymatic degradation and retain sufficientstrength to complete its intended therapeutic role, yet allow thebioprosthetic device to ultimately dissolve, thereby avoiding apermanent implant.

One method of forming, for example, a partially crosslinked skinsubstitute begins with a collagen-based material such as porcinepericardium that is first cleaned of foreign material. Then thecollagen-based material is treated with a generic detergent solution toremove the animal cellular material, leaving behind principally theextracellular matrix consisting almost entirely of type 1 collagen. Theextracellular matrix is then mounted on a frame and soaked for about 50to about 150 hours in the crosslinking reaction agent, such as BDDGE,that has been diluted to the desired concentration (ranging from about1% to about 10%). During the crosslinking process, the temperature ofthe crosslinking reaction agent is maintained at a set temperature(ranging from about −50° C. to about 100° C.), or at differenttemperatures at pre-programmed intervals. At the completion of thecrosslinking reaction, the extracellular matrix is removed from thecrosslinking reaction agent, thoroughly rinsed with water to remove thecrosslinking agent, then dried, packaged, sterilized.

Crosslinked Collagen-Based Material

Some embodiments provide a crosslinked collagen-based materialcomprising Crosslink A and Crosslink B:

where

indicates collagen strands; R¹ from Crosslink A is further defined as

and R² from Crosslink B is further defined as

R³ and R⁴ are independently selected from the group consisting of—(CH₂)_(n)— and —(O(CH₂)_(n))_(m)—, where n and m are independently aninteger from 0-6; and the amount of free amines (—NH₂) on the collagenstrands is between 50% and 85% (or between about 50% and about 85%),relative to the number of free amines on the collagen-based materialbefore any reaction.

In some embodiments, R³ and R⁴ are the same. In some embodiments, the R¹and R² from above are the same. In some embodiments, the amount of freeamines on the crosslinked collagen-based material ranges from 60% to 75%(or about 60% to about 75%), or from 65% to 70% (or about 65% to about70%), relative to the number of free amines on the collagen-basedmaterial before exposing to any crosslinking agent. In some embodiments,the crosslinked collagen-based material further comprises freecarboxylic acid groups. In some embodiments, the un-reactedcollagen-based material has a first percentage of free amine groups onits collagen strands and the crosslinked collagen-based material has asecond percentage of free amine groups, wherein the second percentage offree amine groups is lower than the first percentage as described below.In some embodiments, the crosslinked collagen-based material has apercentage of amines that is lower than that of native collagen.

In some embodiments, the free amine groups of the tailorably crosslinkedcollagen-based material may be blocked using a blocking agent. Ablocking agent is a mono-functional reactive moiety capable of forming astable covalent bond with a free amine or carboxyl groups in theproteins, thereby blocking crosslinking. Typical blocking agents includecompounds containing one amine group or one carboxylic acid group, toblock free carboxylic acids and free amines respectively.

In some embodiments, the ratio of Crosslink A to Crosslink B on thecrosslinked collagen-based material ranges from 100:1 to 1:100 (or about100:1 to about 1:100). In some embodiments, the ratio of Crosslink A toCrosslink B is from 90:10 to 10:90 (or about 90:10 to about 10:90),80:20 to 20:80 (or about 80:20 to about 20:80), from 70:30 to 30:70 (orabout 70:30 to about 30:70), from 60:40 to 40:60 (or about 60:40 toabout 40:60), or 1:1 (or about 1:1).

Bioprosthesis

Some embodiments provide a degradable bioprosthesis synthesized usingany of the methods described above. In some embodiments, the degradablebioprosthesis comprises a crosslinked collagen-based material comprisingCrosslink A and Crosslink B:

where

indicates collagen strands; R¹ from Crosslink A is further defined as

and R² from Crosslink B is further defined as

R³ and R⁴ are independently selected from the group consisting of—(CH₂)_(n)— and —(O(CH₂)_(n))_(m)—, where n and m are independently aninteger from 0-6; and the amount of free amines (—NH₂) on the collagenstrands is between 50% and 85% (or between about 50% and about 85%),relative to the number of free amines on the collagen-based materialbefore any reaction.

In some embodiments, R³ and R⁴ are the same. In some embodiments, the R¹and R² from above are the same. In some embodiments, the amount of freeamines on the degradable bioprosthesis ranges ranges from 60% to 75% (orabout 60% to about 75%), or from 65% to 70% (or about 65% to about 70%),relative to the number of free amines on the collagen-based materialbefore exposing to any crosslinking agent.

In some embodiments, the ratio of Crosslink A to Crosslink B on thedegradable bioprosthesis ranges from 100:1 to 1:100 (or about 100:1 toabout 1:100). In some embodiments, the ratio of Crosslink A to CrosslinkB is from 90:10 to 10:90 (or about 90:10 to about 10:90), 80:20 to 20:80(or about 80:20 to about 20:80), from 70:30 to 30:70 (or about 70:30 toabout 30:70), from 60:40 to 40:60 (or about 60:40 to about 40:60), or1:1 (or about 1:1).

In some embodiments, the degradable bioprosthesis comprises a tailorablycrosslinked collagen-based material as described above, wherein thetailorably crosslinked collagen-based material comprises amine-basedcrosslinks and ester-based crosslinks in the ratios described above. Insome embodiments, as described above, the degradable bioprosthesisfurther comprises free amines.

In some embodiments, the degradable bioprosthesis has a degradation rateof between about 0.2% to about 1.0% per hour when measured using thepronase digestion assay described in the EXAMPLES section. In someembodiments, the degradable bioprosthesis has a degradation rate thatranges from 0.1% to 1.10% (or about 0.1% to about 1.10%) per hour, from0.3% to 1.0% (or about 0.3% to about 1.0%) per hour, from 0.4% to 0.9%(or about 0.4% to about 0.9%) per hour, from 0.5% to 0.8% (or about 0.5%to about 0.8%) per hour, from 0.6% to 0.7% (or about 0.6% to about 0.7%)per hour, from 0.2% to 0.3% (or about 0.2% to about 0.3%) per hour, from0.3% to 0.4% (or about 0.3% to about 0.4%) per hour, from 0.4% to 0.5%(or about 0.4% to about 0.5%) per hour, from 0.5% to 0.6% (or about 0.5%to about 0.6%) per hour, from 0.6% to 0.7% (or about 0.6% to about 0.7%per hour), from 0.7% to 0.8% (or about 0.7% to about 0.8%) per hour,from 0.8% to 0.9% (or about 0.8% to about 0.9%) per hour, from 0.9% to1.0% (or about 0.9% to about 1.0% per hour), or from 1.0% to 1.1% (orabout 1.0% to about 1.1% per hour). It will be appreciated by one ofordinary skill in the art that pronase degradation, enzyme-baseddegradation, and protease-based degradation test results will havevariable degradation rates depending on the type of test used and/or theconditions used during testing.

Heating collagen will induce a structural transition of the nativetriple helical structure at a certain temperature dependent on thenature and degree of crosslinking. Introduction of covalent crosslinkswill increase the stability of the triple helix, thus increasing thedenaturation temperature. The temperature at which denaturation takesplace is also often referred to as shrinkage temperature (Ts), asshrinkage is the macroscopic manifestation of the transformation of thenative triple helix structure to the random coil configuration. Thus, Tscan be used to measure the level of crosslinking. In some embodiments,the Ts of the degradable bioprosthesis is higher than that of nativecollagen. In some embodiments, the Ts of the degradable bioprosthesis isabove 70° C. (or about 70° C.). In some embodiments, the degradablebioprosthesis has a Ts of above 75° C. (or about 75° C.), above 77° C.(or about 77° C.), or above 78° C. (or about 78° C.).

In some embodiments, the amount of free amines is related to thestructural properties of the degradable bioprosthesis. In someembodiments, the amount of free amines on the degradable bioprosthesishas the ranges disclosed above. In some embodiments, the amount of freeamines on the degradable bioprosthesis is proportional to thedegradation rate of the degradable bioprosthesis. In some embodiments,the amount of free amines remaining on the degradable bioprosthesis isproportional to the shrinkage temperature, or denaturation temperatureof the degradable bioprosthesis.

In some embodiments, the degradable bioprosthesis as described herein isformed into a flexible sheet of any shape and from 1 to 500 (or about 1to about 500) square centimeters to be applied to the body. For example,a flexible sheet may be appropriately sized to be placed over treatmentsite. In some embodiments, the flexible sheet may be treated to includean antimicrobial agent to help prevent infection in the sheet or in thetreatment site, and/or to deliver an antimicrobial agent into thetreatment site to treat an existing infection. In some embodiments, themethods described above can be utilized to form a partially crosslinkedskin substitute. The skin substitute may be a non-human tissue such asporcine tissue. The skin substitute may be processed such that thecollagen-containing porcine tissue is only partially cross-linked, andfor example, may be only 20%-80% (or about 20% to about 80%)crosslinked. In another embodiment, the collagen-containing tissue maybe only 40%-60% (or about 40% to about 60%) crosslinked. Thecrosslinking of the tissue may be accomplished using the techniquesdescribed in U.S. Pat. No. 5,880,242, in combination with methods forachieving partial crosslinking as described above.

Method of Treating

Some embodiments provide a method for treating a tissue defectcomprising positioning any of the degradable bioprostheses describedherein at, over, or into the tissue defect. In some embodiments, thetissue defect is a wound. Some embodiments provide a method for treatinga wound, for performing tissue repair, and/or for providing tissue andorgan supplementation. In some embodiments, the first step of treating atissue defect, wound, and/or supplementing and replacing tissue involvesidentifying a patient in need of a degradable bioprosthesis to aid inthe remedying of a tissue defect, healing of a wound, or in need of atissue supplement.

A non-limiting list of patients in need of a degradable bioprosthesisincludes patients suffering tissue defects. In some embodiments, thepatients in need of a degradable bioprosthesis suffer from woundsincluding those in need of skin substitutes for burns and skin ulcers.Degradable bioprostheses can also be used in the treatment of diabeticfoot ulcers, venous leg ulcers, pressure ulcers, amputation sites, inother skin trauma, or in the treatment of other wounds or ailments.Patients in need of a degradable bioprosthesis also include patients inneed of repair and supplementation of tendons, ligaments, fascia, anddura mater. Degradable prostheses can be used in supplement tissue inprocedures including, but not limited to, rotator cuff repair, Achillestendon repair, leg or arm tendon or ligament repair (e.g., torn ACL),vaginal prolapse repair, bladder slings for urinary incontinence, breastreconstruction following surgery, hernia repair, staple or suture linereinforcement, bariatric surgery repair, pelvic floor reconstruction,dural repair, gum repair, and bone grafting and reconstruction. Further,a patient in need of a degradable bioprosthesis also includes one inneed of tissue or organ replacement. In some embodiments, the tailorablycrosslinked collagen-based material described herein can be used toreplace tissue or organs by acting as an artificial extracellularmatrix. In such an application, this tailorably crosslinkedcollagen-based material can be used to support cell and tissue growth.Briefly, cells can be taken from a patient or a viable host and seededon the tailorably crosslinked collagen-based material either in vivo orex vivo. Then as the patient's natural tissues invade the crosslinkedmaterial, it is tailored to degrade and leave only naturally occurringtissues and cells. Other uses for the products and methods describedherein may be for orthopedic surgery and for hernia repair, breast orother soft tissue reconstruction and urogynecological repair.

Skin substitutes or other materials or products made from the processingdescribed above may be used in the treatment of topical and surgicalchronic wounds, or in surgical reconstructive procedures. Whenever humanor animal tissue (biologic products) is used in treating wounds, thebody's normal immunological response is to recognize the foreign tissueand launch an enzymatic attack to degrade/dissolve the tissue collagenas rapidly as possible. Long ago it was shown that crosslinking thecollagen molecules in biologic tissue could mask from the host body theforeign collagen and protect the foreign collagen from enzymatic attack.

In some embodiments, the treatment of an amputation site is performedusing the degradable bioprosthesis. After amputation the surgicallycreated stump commonly has problems healing due pressure and abrasionduring healing process as well as adapting to artificial limbs. In someembodiments, the biodegradable bioprosthesis can be used to aid in thehealing of these wounds and protecting them during the healing process.

The rotator cuff is a group of muscles and their tendons that act tostabilize the shoulder. In some embodiments, the degradablebioprosthesis can be used to augment, reinforce, or replace rotatorcuffs with common tears or ruptures. Rotator cuff tears are commonsports injures and age related degenerative problems of the shoulder. Insome embodiments, the degradable bioprosthesis can function as anadhesion barrier; adhesions are a common post-operative problem withrepairs of this tendinous structure. In some embodiments, common tearsor ruptures of the Achilles tendon can be augmented, reinforced orreplaced with degradable bioprostheses. In some embodiments, thedegradable bioprosthesis can also function as an adhesion barrier.Adhesions are a common problem with repairs of this tendon due to thelong sliding action required as the foot goes through its normal rangeof motion. In some embodiments, the degradable bioprosthesis can be usedto augment, reinforce, or replace tendons and ligaments from the leg,hand, arm, or any other body part.

In some embodiments, the degradable bioprosthesis provides additionalnecessary structural material and a biological material eliminating theneed for permanent synthetic materials being left in the abdomen duringvaginal prolapse treatment. Vaginal vault prolapse is a defect thatoccurs high in the vagina and entails a surgical approach through thevagina or abdomen. The surgical correction of this condition usuallyinvolves a technique called a vaginal vault suspension, in which thesurgeon attaches the vagina to strong tissue in the pelvis or to a bonecalled the sacrum, which is located at the base of the spine. In someembodiments, the degradable bioprosthesis can be used to provideadditional support for the suspension in the treatment of vaginalprolapse.

In some embodiments, the degradable bioprosthesis can be used as abladder sling (or a pubovaginal fascial sling) in the treatment ofurinary incontinence, During this type of operation the urologistattaches a piece of fascia (flat, tough, tendon-like material—about 1inch wide and 5 inches long) around the bladder neck to keep urine in,even under stress. The slings are commonly made of human tissue fromeither the patient or a donor. In some embodiments, the degradablebioprosthesis can replace or augment the human donor material for thisprocedure. The sub urethral sling is commonly made of a synthetic meshplaced under the urethra which acts like a hammock, lifting andcompressing the urethra, preventing leaks. In some embodiment, thedegradable bioprosthesis can replace or augment this synthetic mesh.

In some embodiments, after mastectomy or traumatic injury to the breast,the degradable bioprosthesis can be used to repair or reconstructmissing structural collagen fibers, tendons and fascia during plasticsurgery procedures.

In some embodiments, the degradable bioprosthesis can be used toreinforce or replace the surgical repair and reconstruction of theabdominal wall, fill gaps where insufficient tissue is remainingproviding a scaffold for biologic remodeling, and promote in growthduring hernia repair. Hernia results from the physical failure of theabdominal fascia and muscular structure allowing the protrusion of thecontents of the body cavity. Adhesions are common in hernia. In someembodiments, the degradable bioprosthesis, can also act as an adhesionbarrier post-operatively.

Suture lines, especially in more friable tissues such as lung tissue,weakened or degenerative tendendous materials, heart surgery, organtransplant to name a few require a pledget or suture reinforcement toprevent the suture or staple from “cheese wiring” or cutting through thethin or weakened tissue. In some embodiments, the degradablebioprosthesis can be used as a buttress to prevent cutting through ofthe suture or staple protecting the native tissue.

In some embodiments, the degradable bioprosthesis can be used in placeor in conjunction with sutures or staples to provide a stablereconstruction of the stomach outer fascia and perimeter duringbariatric surgery. Bariatric surgery to reduce the volume of the stomachis commonly done as a remedy for obesity.

In some embodiments, the degradable bioprosthesis can be used toreconstruct and support the weakened and damaged native connectivetissue during pelvic floor reconstruction. Pelvic floor reconstructivesurgery consists of several procedures for correcting a condition calledpelvic organ prolapse. When the muscles of the pelvic floor are damagedor become weak—often due to childbirth—they are sometimes unable tosupport the weight of some or all of the pelvic and abdominal organs. Ifthis occurs, one or more of the organs may drop (prolapse) below theirnormal positions, causing symptoms including discomfort, pain, pressureand urinary incontinence. The goal of pelvic floor reconstruction is torestore the normal structure and function of the female pelvic organs.

In some embodiments, after traumatic injury or brain surgery, thedegradable bioprosthesis can be used to reseal or patch any perforationsand leaks due to defect to the dura mater. Dura mater, the protectivefascia covering of the brain is critical to maintain the fluidsurrounding the brain.

In some embodiments, during the process of repairing damaged or diseasebone segments, the degradable bioprosthesis can be surgically modeled torecreate missing segments or gaps in the repair or replacement ofdamaged bone throughout the skeletal structure. The collagen in corticalbone can retrieved from animal or human bone sourced material. Theprocess of first demineralizing the bone and then cross-linking theremaining collagen structure can result in a material the can functionas a semisolid bone void filler.

In some embodiments, the degradable bioprosthesis can provide analternate source of material gum disease procedures. As a result ofperiodontal disease where gum tissue has been lost, the dentist orperiodontist can surgically insert a soft tissue graft, in whichsynthetic material or tissue taken from another area of your mouth isused to cover exposed tooth roots. In some embodiments, the degradablebioprosthesis can be used to replace or supplement other materials usedin the treatment of periodontal disease.

In some embodiments, after identifying a viable patient for the abovemethods of treating, the next step is to identify the rate at which thedegradable bioprosthesis should degrade in order to work effectively. Insome embodiments, this step involves determining the healing rate of thewound or other tissue defect. In some embodiments, this step involvesdetermining the rate at which natural cell growth would reach a level atwhich the degradable bioprosthesis was no longer necessary.

Advantageously, skin substitute, wound, or other tissue defect treatmentproducts made from the processes described above may be synchronized todegrade with the wound healing process. For example, a skin substitutemade using the processes described herein may be tailored to begin todegrade at more or less a slow, but constant rate, that is synchronizedwith the healing process and the reduction in size of the wound, suchthat upon completion of wound healing, the crosslinked bioprostheticproduct has been fully dissolved, with such process typically completedin 6-12 (or about 6 to about 12) weeks. In some embodiments, thedegradation rate of the skin substitute is such that the skin substitutedegrades completely within the range of time between about 8 weeks andabout 1 year.

Other examples, such as surgical meshes, are made using the processesdescribed herein and may be tailored to resist, or limit degradation,and thereby retain high tensile strength, for a period of time such as1-3 (or about 1 to about 3) months or until such time as thesurrounding, or new tissue has healed sufficiently to bear weight orabsorb normal stresses without damage, after which the product continuesto degrade at a slow, but constant, rate until it is fully dissolved,with such process typically completed in one year or about one year.

Depending on the rate of healing of the wound, a degradablebioprosthesis is then selected for use.

The final step involves implanting the degradable bioprosthesis. In someembodiments, this involves placing the degradable bioprosthesis at,over, or into the tissue defect. In some embodiments, the degradablebioprosthesis is surgically implanted into the patient at the targetarea. In some embodiments, the degradable bioprosthesis is surgicallyimplanted into or over the wound. In some embodiments, the degradablebioprosthesis is affixed into or over the wound with stitches, glue,staples, or other adhesives. In some embodiments, the degradablebioprosthesis is first seeded with autologous or homologous cells beforeimplantation.

In some embodiments, a partially crosslinked material or tailorablycrosslinked collagen-based material, or any degradable bioprosthesis asdescribed herein is formed into a flexible sheet of any shape and from 1to 500 (or about 1 to about 500) square centimeters to be applied to thebody. For example, a flexible sheet may be appropriately sized to beplaced over treatment site. In some embodiments, the flexible sheet maybe treated to include an antimicrobial agent to help prevent infectionin the sheet or in the treatment site, and/or to deliver anantimicrobial agent into the treatment site to treat an existinginfection.

In some embodiments, the product may be sterilized using varioussterilization methods, including ethylene oxide, gamma, steam ande-beam. In some embodiments, a biologic skin substitute or other productmay be sterilized in a manner that minimizes or prevents denaturing ofcollagen, such as by controlling the number of cycles, time, temperatureand dose of radiation.

EXAMPLES

Materials and Equipment:

The following is a list of materials and equipment used throughout theEXAMPLES section: Perkin Elmer Model DSC 4000, Differential Scanningcalorimeter; Boekel Scientific Oven Model 132000; Mettler ToledoAnalytic Balance Model AL54; Mettler Toledo Balance New Classic ML;Calipers, Mitutoyo Corp., 505-626 Dial Caliper; Labconco LyophilizerModel Freezone 6 with Tray Dryer; Scalpel, Bard-parker Stainless SteelSterile blade #10; Sklar Tru-Punch, Disposable Biopsy Punch; VWRSympHony SB70P pH Meter; Distilled Water; Sodium Dodecyl SulfateSolution 0.1%; Sonic Bath=Bransonic 2510; Equine Pericardium; 0.9% NaClSolution; 1,4 Butanediol Digylcidyl Ether; Standard HEPES Buffer; 0.1MPhosphate Buffer pH 4.5 (purchased from Teknova; Cat. #: P4000);Fixation Buffer pH 9.2.

Measuring Free Amine Content:

In addition to the tests described below, amine content can also becalculated. In some embodiments, the free amine group content oftailorably crosslinked collagen-based material, expressed as apercentage of the collagen-based material (%), can be determined using a2,4,6-trinitrobenzenesulfonic acid (TNBS; 1.0 M solution in water,Fluka, Buchs, Switzerland) colorimetric assay. To a sample of 2-4milligrams (mg) of tailorably crosslinked collagen-based material asolution of 1 ml of a 4% (weight/volume) aqueous NaHCO₃ (pH 9.0;Aldrich, Bornem, Belgium) solution and 1 ml of a freshly prepared 0.5%(weight/volume) aqueous TNBS solution can be added. After reaction for 2hours at 40° C., 3.0 ml of 6 M HCl (Merck, Darmstadt, Germany) can beadded and the temperature can be raised to 60° C. When completesolubilization of tailorably crosslinked collagen-based material isachieved, the resulting solution is diluted with 15 ml of deionizedwater and the absorbance was measured on a Hewlett-Packard HP8452AUV/VIS spectrophotometer at a wavelength of 345 nm. A control isprepared applying the same procedure except that HCl was added beforethe addition of TNBS. The free amine group content is calculated using amolar absorption coefficient of 14600 1 mol⁻¹ cm⁻¹ for trinitrophenyllysine [Wang C. L., et al., Biochim. Biophys. Acta, 544, 555-567,(1978)].

The free amine group content of tailorably crosslinked collagen-basedmaterial also can be determined using a ninhydrin test. The followingdescribes the general procedures for testing the amine content of acollagen-based material. Briefly, a sample of 1-25 milligrams (mg) oftailorably crosslinked collagen-based material is collected. Next, asolution of 1 ml of a 4% (weight/volume) ninhydrin in methyl cello solveis prepared. Then a 0.2 M sodium citrate buffer is prepared bydissolving 1.05 g of citric acid monohydrate and 0.04 g of stannouschloride dihydrate in 11 mL of 1.0 N NaOH and adding 14 mL of purifiedwater. The pH of the sodium citrate buffer is adjusted to 4.9 to 5.1with HCl and/or NaOH. Next, the 4% ninhydrin solution and sodium citratebuffer are mixed in a dark bottle for immediate use. Now a solution ofN-acetyllysine (ALys) is prepared by disolveing 47.1 mg of ALys in 50 mLof purified water. The ALys is used as a standard solution forcalibrating the absorbance which is read at 570 nm. After a standardcurve is plotted, samples of dried tissue are tested. Each solution forto be read by absorbance is prepared using 1 mL of buffered ninhydrin,100 microliters of purified water, and the tissue or control sample. Thetest solutions are heated to 100° C. for 20 minutes, cooled, then 5 mLof isopropyl alcohol is added. The absorbance is then read and theamount of mols of amine per gram of sample and control is calculatedfrom using the following equation: A=mX+b where, A=absorbance, X=contentof ALys in micromoles, m=the slope, and b=the y-intercept. The contentof micromoles of free amine in the sample is thenX_(samp)=(A_(samp)−b)/m.

Mechanical Properties:

Stress-strain curves of the degradable bioprosthesis can be taken usinguniaxial measurements using a mechanical tester. Tensile bars (40.0mm×4.0 mm×1.4 mm) can be cut using a dumb-bell shaped knife and can behydrated for at least one hour in PBS at room temperature. The thicknessof the samples can be measured in triplicate using a spring-loaded typemicrometer (Mitutoyo, Tokyo, Japan). An initial gauge length of 10 mmwas used and a crosshead speed of 5 mm/minute can be applied untilrupture of the test specimen occurs. A preload of 0.05 N can be appliedto prestretch the specimen before the real measurement. The tensilestrength, the elongation at alignment, the elongation at break, the lowstrain modulus and the high strain modulus of the sample can becalculated from five independent measurements.

Preparation of Pericardium to Form Collagen-Based Material:

Equine pericardial sacks were procured fresh from Carnicos de Jerez S.A. de C. V. and air freighted in 0.9% NaCl solution on ice. Immediatelyon receipt, all sacks were rinsed in fresh, cold 0.9% NaCl solution,debrided of fat and excess fibrous tissue, and trimmed with a surgicalscalpel to create 8 similar patches approximately 10 cm×15 cm. Allpatches were decellularized by a process of 20 minutes sonication in a0.1% solution of Sodium Dodecyl Sulfate (SDS) followed by three separaterinses in 500 ml of 0.9% NaCl solution to remove excess SDS. Thedecellularization process is intended to remove any excess intracellularmaterials. The anionic surfactant (SDS) used in the process also helpsto reduce excess fats and oils. The treatment of the resulting patchesyielded 8 debrided, decellularized pericardial patches. One of thesepatches was set aside as a control for crosslinking experiments.

Preparation of Crosslinking Solutions:

To 1 L of deionized water was added potassium carbonate (6.5 grams) andsodium bicarbonate (16.6 grams). The solution was mixed until all solidsdissolved. The pH of the solution was measured using a pH meter whereinthe target pH was 9.2±0.2. If necessary, the pH of the solution wasadjusted to 9.2±0.2 by adding dilute NaOH or dilute HCl. Next, to thebuffered solution (bicarbonate buffer) was added BDDGE (40 g) to afforda 4% by weight solution of BDDGE. This solution was stirred to give ahomogenous solution of 9.2±0.2 pH BDDGE.

To a solution phosphate buffered solution (PBS, 0.5 L, 4.5±0.2 pH) wasadded BDDGE (20 g) to afford a 4% by weight solution of BDDGE. Thissolution was stirred to yield a homogeneous solution of 4.5±0.2 pHBDDGE.

Example 1

To a low pH solution of BDDGE (4.0% w/v BDDGE, 4.5±0.2 pH, excess) wasadded, an approximately 10×15 cm debrided, decellularized pericardialpatch. The patch was allowed to remain in the BDDGE/PBS solution for 160hours at which time the patch was rinsed with distilled waterthoroughly.

Example 2

To a high pH solution of BDDGE (4.0% w/v BDDGE, 9.2±0.2 pH, excess) wasadded, an approximately 10×15 cm debrided, decellularized pericardialpatch. The patch was allowed to remain in the 4.0% w/v BDDGE, 9.2±0.2 pHsolution for 8 hours, at which time it was added to a low pH solution ofBDDGE (4.0% w/v BDDGE, 4.5±0.2 pH, excess). After 152 hours, the patchwas removed from the low pH BDDGE solution and was rinsed with distilledwater thoroughly.

Example 3

To a high pH solution of BDDGE (4.0% w/v BDDGE, 9.2±0.2 pH, excess) wasadded, an approximately 10×15 cm debrided, decellularized pericardialpatch. The patch was allowed to remain in the 4.0% w/v BDDGE, 9.2±0.2 pHsolution for 24 hours, at which time it was added to a low pH solutionof BDDGE (4.0% w/v BDDGE, 4.5±0.2 pH, excess). After 136 hours, thepatch was removed from the low pH BDDGE solution and was rinsed withdistilled water thoroughly.

Example 4

To a high pH solution of BDDGE (4.0% w/v BDDGE, 9.2±0.2 pH, excess) wasadded, an approximately 10×15 cm debrided, decellularized pericardialpatch. The patch was allowed to remain in the 4.0% w/v BDDGE, 9.2±0.2 pHsolution for 36 hours, at which time it was added to a low pH solutionof BDDGE (4.0% w/v BDDGE, 4.5±0.2 pH, excess). After 124 hours, thepatch was removed from the low pH BDDGE solution and was rinsed withdistilled water thoroughly.

Example 5

To a high pH solution of BDDGE (4.0% w/v BDDGE, 9.2±0.2 pH, excess) wasadded, an approximately 10×15 cm debrided, decellularized pericardialpatch. The patch was allowed to remain in the 4.0% w/v BDDGE, 9.2±0.2 pHsolution for 48 hours, at which time it was added to a low pH solutionof BDDGE (4.0% w/v BDDGE, 4.5±0.2 pH, excess). After 112 hours, thepatch was removed from the low pH BDDGE solution and was rinsed withdistilled water thoroughly.

Example 6

To a high pH solution of BDDGE (4.0% w/v BDDGE, 9.2±0.2 pH, excess) wasadded, an approximately 10×15 cm debrided, decellularized pericardialpatch. The patch was allowed to remain in the 4.0% w/v BDDGE, 9.2±0.2 pHsolution for 64 hours, at which time it was added to a low pH solutionof BDDGE (4.0% w/v BDDGE, 4.5±0.2 pH, excess). After 96 hours, the patchwas removed from the low pH BDDGE solution and was rinsed with distilledwater thoroughly.

Example 7

To a high pH solution of BDDGE (4.0% w/v BDDGE, 9.2±0.2 pH, excess) wasadded, an approximately 10×15 cm debrided, decellularized pericardialpatch. The patch was allowed to remain in the 4.0% w/v BDDGE, 9.2±0.2 pHsolution for 160 hours, at which time the patch was removed from the lowpH BDDGE solution and was rinsed with was rinsed with distilled waterthoroughly. FIG. 2 depicts a sheet of collagen-based materialcrosslinked for 160 hours at pH 9.2 with BDDGE. Each of the collagenmaterials from Examples 1-6 are identical in appearance to the FIG. 2material when viewed with the naked eye.

Temperature of Shrinkage (Ts):

Three 3 mm diameter samples were cut from each of the resultingcrosslinked materials from examples 1-7 and the control using a Skylar 3mm biopsy punch.

Each sample was sealed in a Perkin Elmer DSC volatile sample pan(0219-0062). An empty pan is run in parallel with the test sample in theDifferential Scanning calorimeter (DSC). Through comparison of heat flowof the empty pan and test pan, the peak temperature of enthalpyindicates the transition temperature or temperature of shrinkage (Ts) ofthe sample expressed in ° C.

Ts of the samples are compared to that of the control ornon-cross-linked to determine the comparative level of aminecross-linking present. Table 1 contains the results of each sample,separated by each example number. FIG. 3 contains a graphical depictionof the results from Table 1.

TABLE 1 Temperature of Shrinkage Results (° C.) Sample # 1 2 3 Ave SDExample 1 69.63 70.90 69.71 70.08 0.58 Example 2 72.81 72.40 72.41 72.540.19 Example 3 73.91 74.09 74.19 74.06 0.12 Example 4 76.17 75.98 76.0376.06 0.08 Example 5 78.31 78.54 78.53 78.46 0.11 Example 6 77.58 77.1176.98 77.22 0.26 Example 7 77.26 77.30 78.21 77.59 0.44 Control 69.1968.94 68.55 68.89 0.26Pronase Digestion Assay:

Three 1 cm×1 cm samples were cut from each of examples 1-7 and thecontrol and tested per MF3-00X Pronase Digestion. Per procedure MF3-00Xeach sample was placed in a 5 ml glass scintillation vial with 4 mls ofa HEPES buffered solution with 95 mg/100 ml bacterial protease derivedfrom Streptomyces griseus.

The samples were incubated at 45° C. for 24 hours, blotted dry andlyophilized in the Labconco lyophilizer. Then each sample was weighedutilizing the Mettler Toledo analytic balance. All samples werereweighed using the Mettler Toledo analytic balance.

The percent degradation was determined calculating the percent change inweight before and after 24 hours exposure to the protease. Table 2contains the results of each sample, separated by each example number.FIG. 4 contains a graphical depiction of the results from Table 2.

TABLE 2 Protease Digestion % After 24 Hours Exposure to Protease Sample# Wt. (mg) after/ Ave % before 1 2 3 Remaining SD Example 1 5.30/7.605.40/7.30 5.60/7.30 73.47% 2.87 Example 2  7.80/10.10  8.30/10.5010.60/13.20 78.86% 1.26 Example 3 10.60/11.90 10.80/12.20 12.40/13.8089.15% 0.55 Example 4 10.50/11.90 11.30/12.90 12.70/13.90 89.07% 1.65Example 5 14.30/14.80 15.70/16.00 19.80/20.50 97.11% 0.72 Example 65.00/5.40 6.10/6.40 5.10/5.50 93.54% 1.25 Example 7 11.50/12.0012.10/12.60 12.60/12.90 96.51% 0.83 Control  8.60/13.40  8.80/13.40 9.40/13.50 66.49% 2.30

The degradation rate was then calculated by dividing the % digestionafter 24 hours by 24 hours to yield % degraded/hour. Table 3 shows thoseresults. FIG. 5 contains a graphical depiction of the results from Table3.

TABLE 3 Degradation Rate in %/hr. Sample # 1 2 3 Ave SD Example 1 1.261.08 0.97 1.11 0.15 Example 2 0.95 0.87 0.82 0.88 0.06 Example 3 0.460.48 0.42 0.45 0.03 Example 4 0.49 0.52 0.36 0.46 0.08 Example 5 0.140.08 0.14 0.12 0.04 Example 6 0.31 0.20 0.30 0.27 0.06 Example 7 0.170.17 0.10 0.15 0.04 Control 1.49 1.43 1.27 1.40 0.12

The study demonstrated that a pH shift from high (9.2) to low (4.5)within the first 64 hours of a 160 hour 4% BDDGE cross-linking processresulted in an extracellular collagen matrix with progressively lower Tsvalues, lower resistance to protease and a significantly fasterbioresorbtion rate.

The process of pH modulation of the BDDGE cross-linking process ofextracellular collagen matrix material is a feasible method of producinga medical device for general surgical repair with a controlledpredetermined bioresorbtion rate.

Various modifications to the implementations described in thisdisclosure may be readily apparent to those skilled in the art, and thegeneric principles defined herein may be applied to otherimplementations without departing from the spirit or scope of thisdisclosure. Thus, the disclosure is not intended to be limited to theimplementations shown herein, but is to be accorded the widest scopeconsistent with the principles and features disclosed herein. Certainembodiments of the invention are encompassed in the claim set listedbelow.

What is claimed is:
 1. A crosslinked collagen-based material comprisingCrosslink A and Crosslink B:

wherein:

indicates collagen; R¹ is

R² is

R³ and R⁴ are each —CH₂—O—(CH₂)_(n)—O—CH₂— and n is 4; wherein thecrosslinked collagen based material has a degradation rate between about0.2% to about 1.0% per hour during the first 24 hours of exposure to apronase digestion assay.
 2. The crosslinked collagen-based material ofclaim 1, wherein the amount of free amines (—NH₂) on the collagen isbetween 50% and 85% relative to the total amount of free amines (—NH₂)prior to crosslinking.
 3. The crosslinked collagen-based material ofclaim 1, wherein the ratio of Crosslink A and Crosslink B is from about80:20 to about 20:80.
 4. The crosslinked collagen-based material ofclaim 1, wherein the ratio of Crosslink A and Crosslink B is from about70:30 to about 30:70.
 5. The crosslinked collagen-based material ofclaim 1, wherein the ratio of Crosslink A and Crosslink B is from about100:1 to about 1:100.
 6. A degradable bioprosthesis, comprising: acrosslinked collagen-based material comprising Crosslink A, Crosslink B,and free amines (—NH₂):

wherein:

indicates collagen; R¹ is

R² is

R³ and R⁴ are each —CH₂—O—(CH₂)_(n)—O—CH₂— and n is 4; wherein thecrosslinked collagen based material has a degradation rate between about0.2% to about 1.0% per hour during the first 24 hours of exposure to apronase digestion assay.
 7. The crosslinked collagen-based material ofclaim 1, wherein the amount of free amines (—NH₂) on the collagen isbetween 50% and 85% relative to the total amount of free amines (—NH₂)prior to crosslinking.
 8. The crosslinked collagen-based material ofclaim 1, wherein the ratio of Crosslink A and Crosslink B is from about80:20 to about 20:80.
 9. The crosslinked collagen-based material ofclaim 1, wherein the ratio of Crosslink A and Crosslink B is from about70:30 to about 30:70.
 10. The crosslinked collagen-based material ofclaim 1, wherein the ratio of Crosslink A and Crosslink B is from about100:1 to about 1:100.
 11. A crosslinked collagen-based materialcomprising Crosslink A, Crosslink B, and free amines (—NH₂):

wherein:

indicates collagen strands; R¹ is

R² is

R³ and R⁴ are each —CH₂—O—(CH₂)_(n)—O—CH₂— and n is 4, and the amount offree amines (—NH₂) on the collagen strands is between 50% and 85%relative to the total amount of free amines (—NH₂) prior tocrosslinking, and the ratio of amine-based crosslinks to ester-basedcrosslinks is about 100:1 to about 1:100.
 12. The crosslinkedcollagen-derived material of claim 11, wherein the crosslinkedcollagen-derived material has a degradation rate between about 0.2% toabout 1.1% per hour when subjected to a pronase digestion assay.
 13. Thecrosslinked collagen-based material of claim 11, wherein the collagenstrands are derived from animal pericardium.
 14. The crosslinkedcollagen-based material of claim 11, having a shrinkage temperature (Ts)above about 70° C.
 15. The crosslinked collagen-based material of claim11, wherein the shrinkage temperature is proportional to the amount offree amines on the collagen strands.
 16. The crosslinked collagen-basedmaterial of claim 11, wherein the amount of free amines on the collagenstrands is between about 60% to about 75%.
 17. The crosslinkedcollagen-based material of claim 11, wherein the degradation rate isproportional to the amount of free amines on the collagen strands. 18.The crosslinked collagen-based material of claim 11, wherein thecrosslinked collagen-based material is a flexible sheet.
 19. Thecrosslinked collagen-based material of claim 18, wherein the flexiblesheet is treated with antimicrobial agent.